PROJECT SUMMARY Vibrio parahaemolyticus is a leading cause of seafood-borne diarrheal disease and a multidrug resistant emerging pathogen. Although it is known that one of the type 3 secretion systems (T3SS2) plays an essential role in bacterial colonization and diarrheal disease, the assembly of a functional T3SS2 apparatus and the effectors that are responsible for pathological alterations during infection are not completely defined. Continued existence of this gap represents an important problem because, until it is filled, the likelihood that pharmacological intervention of V. parahaemolyticus infection by targeting specific virulence factors is remote. The long-term goal is to harness the medical benefits that are offered by defining T3SS2 apparatus assembly and the function of effector proteins during V. parahaemolyticus infection. The overall objective here is to identify proteins that are required for the assembly of functional T3SS2 apparatus and enhancing intestinal cell proliferation. The central hypothesis is that VopI, an essential component for the assembly of functional T3SS2 apparatus, also serves as an effector protein to regulate intestinal cell proliferation for the benefit of intestinal colonization. This hypothesis has been formulated on the basis of our own preliminary data that deletion of VopI blocked the secretion and translocation of T3SS2 substrates and VopI itself can be translocated by T3SS2 into host cell nucleus to regulate cell proliferation and bacterial colonization. The rationale for the proposed research is that, once the role of VopI in the assembly of T3SS2 apparatus and epithelial cell proliferation is elucidated, both T3SS2 assembly and intestinal epithelial cell proliferation could be pharmacologically modulated, resulting in new and innovative approaches for the prevention and treatment of infection with V. parahaemolyticus. Furthermore, the results obtained from this study will shed new light on the role of T3SS apparatus protein as an effector both in vitro and in vivo during bacterial infection. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Define the role of VopI as a structural component in the assembly of T3SS2 apparatus; 2) Define the mechanisms by which VopI, as an effector, promotes cell proliferation; and 3) Define the role of VopI as an effector in vivo using an infant rabbit model. In the first aim, we will determine VopI localization, its interaction partners within the T3SS2 apparatus and its effect on T3SS2 biogenesis using electron microscopy, confocal microscopy and biochemical approaches. In the second aim, we will elucidate the mechanism of VopI-mediated cell proliferation. Particularly, we will determine the biological significance of the interaction between VopI and a host nucleolar protein, EBP2. In the third aim, we will determine the role of VopI, as an effector, in intestinal cell proliferation in vivo and the contribution of cell proliferation to bacterial colonization and virulence.